Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
  • C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J23/00—Details of transit-time tubes of the types covered by group H01J25/00
  • H01J23/16—Circuit elements, having distributed capacitance and inductance, structurally associated with the tube and interacting with the discharge
  • H01J23/24—Slow-wave structures, e.g. delay systems
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J25/00—Transit-time tubes, e.g. klystrons, travelling-wave tubes, magnetrons
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J25/00—Transit-time tubes, e.g. klystrons, travelling-wave tubes, magnetrons
  • H01J25/02—Tubes with electron stream modulated in velocity or density in a modulator zone and thereafter giving up energy in an inducing zone, the zones being associated with one or more resonators
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/814—Enzyme separation or purification

Definitions

• the present invention is concerned with a process for the enrichment of proteins.
• the expression enrichment of proteins is intended to embrace processes, such as precipitation, purification and fractionation of proteins, which result in altering the concentration, purity, or type of protein between un-enriched and enriched mixtures.
• agents and processes are already known for the enrichment of proteins, especially proteins from biological material.
• agents for this purpose include ammonium sulfate, inorganic gels and activated charcoal and examples of processes for to effect enrichment include chromatography on exchangers and mo lecular sieves.
• the present invention provides a process for the enrichment of dissolved proteins, especially of enzymatically-active proteins, which is flexible andapplicable to different proteins and impurities and thus overcomes the disadvantages of the prior art.
• the process of the invention comprises the use of polyethylene-imine in enrichment processes, as will be detailed below.
• polyethylene-imine As a flocculation adjuvant, for example, for the clarification "ice of waste water, for the improvement; of the filterability of precipitates and the like. Furthermore, it has-.already been suggested to use polyethylene-imine (which'has been rendered insoluble) in the manner of anion exchanger for the chromatography of nucleic acids. .In addition, 'polyethylene-imine-is used -as ;an .adhesiomagent in;.various branchesv of industry, for-example,- for fibres, dyes, adhesives, lacquers and the like. However, a knowledge of these various uses would not suggest that watersoluble polyethylene-imine would be particularly suitable .for the enrichment of dissolvedproteins.
• the present invention therecane-zu sed the various commercially available types of-polyethyleneimine from weakly basic via average ,basic to strongly basic.
• the separation effect which can be achieved depends, to a certain extent, upon the-molecular weight of the polyethylene-imine so that, with certain molecular weights, depending upon the particular protein, especially favorable enrichment effects can often vbe achieved.
• the conventionally modified polyethylene-imin es for example partially or completely ethoxylated polyethylene-imine can also be used.
• the protein solution is mixed with a small amount of polyethylene-imine, whereupon the protein precipitates out.
• the precipitation takes place at a low ion concentration and high dilution with regard to the polyethylene-imine.
• the precipitated protein can again be brought into solution.
• the precipitation is dependent upon the pH range and generally takes place at a pH between 5.0 and 9.0, preferably at a pH between 5.5 and 8.5. Above and below these limiting pH values, precipitated protein .norf mally goes into solution again.
• the separation of the precipitate containing protein and polyethylene-imine can take place according to the methods normally used for the enrichment of proteins.
• the polyethylene-imine can be separated on cation exchangers or, on the contrary, the protein can be separated on anion exchangers.
• Further examples of possible separation methods include ammonium sulfate precipitation, precipitation with organic solvents and molecular sieve separation, especially when the poly% ethylene-imine used has, in comparison with the precipi tated protein, a very large or a very small molecular weight.
• the term enrichment also includes the purification of proteins
• the purification can consist of a precipitation, such as has been described above, in which certainimpuritie's remain behind in a dissolved state.
• a further possibility of purification is a so-called negative precipitation.
• the impurities are first precipitated, whereas the desired pro tein remains in the supernatant and, for example, after separation of the first precipitate, is separately precipitated out.
• ballast materials can beprecipi tated'out, as explained hereinafter in more. d etail,' at high ion concentrations at which the desired protein er proteins remains or remain in solution.
• the term enrichment is also to be regarded as including frac tionation.
• Fractionation is the separation of inactive proteins from desired active proteins.
• Inactive proteins are here to be understood to include proteins which are themselves active but which are not desired in particular cases or which do not have the required activity.
• the fractionation takes place by the addition of increasing amounts of polyethylene-imine, the various proteins thereby precipitating out one after the other with a surprisingly good separation efiFect.
• ballast materials i.e. those materials which, inthe purification step, within the meaning of the present invention, are to be separated from active proteins, there are to be understood nucleic acids, cell wall components, such as polyuronic acids and the like, and plasma components other than the desired proteins themselves.
• An especial advantage of the process according to the present invention is that only very small amounts of polyethylene-imine need to be added in order to be able to achieve the desired separation effects.
• considerably greater amounts of polyethylene-imine solution are to be added, for example, 15-20 volume percent of a 10% solution or even more.
• the process according to the present invention is extraordinarily mild and proceeds, therefore, with the best possible maintenance of the activity of the protein. As the following examples show, in the case of negative enrichment steps, there can be achieved activity yields of up to 100%, whereas in the case of positive enrichment steps, i.e. with precipitation of the particular protein wanted, activity yields of up to 90% can be achieved.
• EXAMPLE 1 Separation of albumin and ribonucleic acid (RNS) by means of polyethylene-imine (PEI) with a molecular weight about 50,000
• 500 mg. serum alubumin (corresponding to 400 mg. protein according to the biuretmethod) and 500 mg. ribonucleic acid were dissolved in 50 ml. 0.05 M phosphate bulfer (pH 7.0) and the pH value then adjusted to 7.0 again with 2 N sodium hydroxide solution.
• polyethylene-imine molecular weight 50,000; 10% solution; pH "7.0
• the albumin fiinally precipitates out (see Table 1).
• Glucoe-6-phosphate-dehydrogenase (G-6-PDH) from Bakers yeast pH 7.0) were then slowly added. A precipitate was formed which contains the nucleic acids and the proteases. The PEI precipitation can also-be carried out directly on the yeast incubation liquid without previous centrifuging.
• the PEI precipitate was coarsely flocculated and can be very readily centrifuged, together with burst yeast cells.
• the enzyme solution (supernatant) pre-purified in this manner was. adjusted with solid ammonium sulfate to 3.05 M, the precipitate obtained containing the two enzymes. After centrifuging, the precipitate was taken up with 2.10' M magnesium chloride solution, containing 1.10- M Trilion and 110- M sodium azide (pH 6.5), and dialysed for 12 hours against tap water.
• the dialysate (about 2.0 liters; 0.01 M ammonium sulfate content) are mixed with 0.0l00.020 volumes of the above PEI solution so that just about 5% of the G-6-PDH remains in the supernatant.
• This operation was carried out at a pH between 6.0 and 7.5, preferably at a pH of 7.0. The supernatant obtained was discarded.
• the PEI-enzyme precipitate was suspended in a 0.01 M phosphate buffer (pH 7.6) and centrifuged; the wash water contains about 2% of the enzyme activity.
• EXAMPLE 3 Isolation of hexokinase (HK) from bakers yeast The process according to Example 2 was repeated up to the production of a dialysate.
• PEI polyethyleneimine
• the enzyme precipitate was taken up with 0.025 M phosphate buffer (pH 7.6), stirred for 15 minutes at ambient temperature and subsequent centrifuged. The supernatant contains about 40% of the HK activity, referred to the dialysate. The purification in this step was about fourfold (see Table '3).
• EXAMPLE 5 Glucose-oxidase from Aspergillus niger 1 kg. pressed mycelia of Aspergillus niger were stirred up with 2 liters completely desalinated water and homogenised at 300-400 ats. by high pressure dispersion.
• the homogenisate was mixed with 0.2 volume percent of a 10% solution of PEI (molecular weight 1800; pH 7.0-7.5) and, after stirring for about 30 minutes, filtered off.
• the clear filtrate contains the glucose oxidase, the ballast materials being in the precipitate which was separated off and discarded.
• the salt-poor supernatant (conductivity at C. about 2 x 10%. Siemens) was mixed with 0.1 volume percent of a 10% PEI solution (molecular weight 30,00060,000) at pH 7.0-7.5. After stirring for about minutes, in the clear-filtered supernatant there remain 8% of the activity of the glucose oxidase.
• 60 g. of the dried mycelia were extracted for 3 hours at 37 C. in 600 ml. 0.05 M dipotassium hydrogen phosphate, 10* M glycerol and 10* M ethylene-diaminatetraacetic acid (EDTA). This was then diluted with 600 ml. 10 M glycerol and 10- M EDTA and the mycelia centrifuged off. The precipitate was discarded.
• the extract obtained was heated at 60 C. for 60 minutes, cooled to 10 C. and denatured protein centrifuged off.
• the supernatant from the heating step was saturated with ammonium sulfate to 3.2 M and the precipitated glycerokinase centrifuged off. The inactive supernatant was discarded. In the precipitate, the glycerokinase was taken up with 0.05 M potassium phosphate buffer (pH 7.6), l0- M glycerol and 10* M EDTA and dialyzed at 0 C. for a total of 14 hours against the same buffer.
• the clear extract was mixed with 0.3 volume percent of a 10% PEI solution (molecular weight 30,000-60,000) at pH 7.0-7.5. After about 30 minutes stirring, the activity of the glycerokinase remains in the clear centrifuged supernatant.
• a 10% PEI solution molecular weight 30,000-60,000
• the precipitate was extracted with 0.05 M potassium phosphate buffer (pH 7.0) and centrifuged. The centrifuged off precipitate was discarded. The glycerokinase in the supernatant was precipitated out by adding ammonium sulfate to 3.2 M. The precipitate was taken up in concentrated form in 0.05 M potassium phosphate buffer (pH 7.0), 10* M glycerol and 10* M EDTA and dialyzed for 14 hours at 0 C. against the same buffer.
• the dialystate was diluted 1:1 with 0.01"M, potassiumphosphate butter (pH 7.0). 0.5. volume percent of a PEI solution (molecular weight 1800; pH adjusted to 7.0 with 2 N sodium hydroxide solution) were then added, the ballast materials thereby precipitating out. The inactive precipitate was centrifuged off and the diaphora'se in the supernatant was frozen and lyophilized. i a Table 7 shows the values obtained with regard ,to yield and enrichment.
• Process for the enrichment of enzymatically active dissolved proteins which comprises treating a mixture containing the dissolved protein with a water soluble polyethylene-imine in aqueous solution wherein said polyethylene-imine has an average molecular weight from about 600 to about 100,000 to precipitate out either the protein or impurities contained in said mixture, and recovering the enriched protein.
• said protein mixture contains plurality of enzymatically active proteins wherein the non-desired proteins are first precipitated out upon addition of the polyethylene-imine, leaving the desired protein in solution.

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• 1970
  • 1970-01-16 DE DE2001902A patent/DE2001902C3/de not_active Expired
  • 1970-10-01 AT AT888770A patent/AT307447B/de not_active IP Right Cessation
  • 1970-10-20 IL IL35497A patent/IL35497A/xx unknown
  • 1970-10-20 JP JP45092317A patent/JPS4939836B1/ja active Pending
  • 1970-11-10 ZA ZA707605A patent/ZA707605B/xx unknown
  • 1970-12-01 NL NLAANVRAGE7017514,A patent/NL168267C/xx not_active IP Right Cessation
  • 1970-12-31 FR FR7047458A patent/FR2075149A5/fr not_active Expired
• 1971
  • 1971-01-11 SE SE00227/71A patent/SE368408B/xx unknown
  • 1971-01-13 CH CH49171A patent/CH560723A5/xx not_active IP Right Cessation
  • 1971-01-14 GB GB0828/71A patent/GB1298431A/en not_active Expired
  • 1971-01-15 DK DK17071A patent/DK145601C/da not_active IP Right Cessation
• 1972
  • 1972-11-13 US US00306124A patent/US3794562A/en not_active Expired - Lifetime

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